U.S. patent application number 11/439288 was filed with the patent office on 2006-11-23 for high throughput multi-antigen microfluidic fluorescence immunoassays.
Invention is credited to W. French Anderson, Emil P. Kartalov, Axel Scherer, Clive Taylor.
Application Number | 20060263818 11/439288 |
Document ID | / |
Family ID | 37448746 |
Filed Date | 2006-11-23 |
United States Patent
Application |
20060263818 |
Kind Code |
A1 |
Scherer; Axel ; et
al. |
November 23, 2006 |
High throughput multi-antigen microfluidic fluorescence
immunoassays
Abstract
The development of a high-throughput multi-antigen microfluidic
fluorescence immunoassay system is illustrated in a 100-chamber
PDMS (polydimethylsiloxane) chip which performs up to 5 tests for
each of 10 samples. Specificity of detection is demonstrated and
calibration curves produced for C-Reactive Protein (CRP), Prostate
Specific Antigen (PSA), ferritin, and Vascular Endothelial Growth
Factor (VEGF). The measurements show sensitivity at and below
levels that are significant in current clinical laboratory practice
(with SIN>8 at as low as 10 pM antigen concentration). The chip
uses 100 nL per sample for all four tests and provides an improved
instrument for use in scientific research and "point-of-care"
testing in medicine.
Inventors: |
Scherer; Axel; (Laguna
Beach, CA) ; Kartalov; Emil P.; (Los Angeles, CA)
; Anderson; W. French; (San Gabriel, CA) ; Taylor;
Clive; (South Pasadena, CA) |
Correspondence
Address: |
Daniel L. Dawes;Myers Dawes Andras & Sherman LLP
19900 MacArthur Boulevard
Irvine
CA
92612
US
|
Family ID: |
37448746 |
Appl. No.: |
11/439288 |
Filed: |
May 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60683822 |
May 23, 2005 |
|
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|
Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/7.1 |
Current CPC
Class: |
B01L 3/50273 20130101;
B01L 3/502707 20130101; B01L 2400/0655 20130101; B01L 2400/0481
20130101; B01L 2400/0487 20130101; B01L 2200/0605 20130101; B01L
2200/0621 20130101; G01N 33/582 20130101; B01L 2300/0861 20130101;
B01L 3/502738 20130101; B01L 2300/0636 20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; C12M 1/34 20060101
C12M001/34 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] Financial support was provided for the invention by the
National Institutes of Health under NIH Grant no. 1R01 HG002644.
The U.S. Government has certain rights to the invention.
Claims
1. A microfluidic assay apparatus comprising: a matrix; a plurality
of sample/buffer flow channels defined in the matrix; a plurality
of antibody/buffer flow channels defined in the matrix and
intersecting the plurality of sample/buffer flow channels; a
corresponding plurality of selectively controllable, valved capture
microchambers, the capture microchamber being defined at each
intersection of the plurality of sample/buffer flow channels and
the plurality of antibody/buffer flow channels; means for
collecting a protein in the plurality of capture microchambers; and
means for detecting the plurality of collected proteins in the
capture microchambers.
2. The microfluidic assay apparatus of claim 1 where the means for
detecting the plurality of collected proteins in the capture
microchambers comprises means for quantifying the concentration of
the protein, which is collected in the capture microchambers.
3. The microfluidic assay apparatus of claim 1 where the means for
detecting the plurality of collected proteins in the capture
microchambers comprises means for qualitatively identifying the
protein, which is collected in the capture microchambers.
4. The microfluidic assay apparatus of claim 1 where means for
collecting the protein in the plurality of capture microchambers
comprises means for simultaneously collecting a plurality of
different kinds of proteins in corresponding different capture
microchambers.
5. The microfluidic assay apparatus of claim 1 where the plurality
of sample/buffer flow channels are arranged and configured to
simultaneously receive a plurality of different samples.
6. The microfluidic assay apparatus of claim 1 where the matrix is
comprised of a selectively epoxide coated substrate and at least
one PDMS layer disposed on the epoxide coated substrate.
7. The microfluidic assay apparatus of claim 6 where each of the
selectively controllable, valved capture microchambers are defined
in the at least one PDMS layer and comprise at least one push-down
or pull-up valve to control flow into or out of the capture
microchamber.
8. The microfluidic assay apparatus of claim 1 where the means for
simultaneously collecting a plurality of different kinds of
proteins in corresponding different capture microchambers comprises
a plurality of antigens.
9. The microfluidic assay apparatus of claim 1 where the plurality
of antibodies are selectively attached to the substrate by means of
selectively epoxide coated substrate surfaces.
10. The microfluidic assay apparatus of claim 1 where ones of the
plurality of sample/buffer flow channels are selectively coupled
through selective communication of at least two controllable,
valved capture microchambers to form a circulation path of fixed
volume and further comprising a pump included in the circulation
path to circulate fluid in the path for a predetermined interval to
increase collection of the protein in the at least two capture
microchambers.
11. The microfluidic assay apparatus of claim 1 where one of the
plurality of sample/buffer flow channels is selectively
communicated by selective valve actuation to a selected capture
microchamber and portion of the communicated sample/buffer flow
channel to form a path of fixed volume and further comprising a
pump included in the path to flow fluid in the path for a
predetermined interval to increase collection of the protein in the
at the selected capture microchamber.
12. The microfluidic assay apparatus of claim 1 where the plurality
of capture microchambers are selectively sized to provide a capture
surface, which is scaled according to an expected concentration of
protein.
13. The microfluidic assay apparatus of claim 12 where the capture
surface is smaller, the lower is the expected concentration of
protein.
14. The microfluidic assay apparatus of claim 1 further comprising
means for diluting a sample with a predetermined amount of buffer
to adjust the sample concentration into an acceptable range of
measurement within the microchambers.
15. A method of performing a microfluidic assay comprising:
selectively flowing selected monoclonal antibodies in a plurality
of horizontal flow channels in a microfluidic, optical transparent,
biologically inert matrix; selectively bonding selected monoclonal
antibodies to binding moieties on a surface in a corresponding
microchambers in the microfluidic matrix; flowing a derivatization
buffer in the horizontal flow channels to remove unbound excess
protein and to passivate any unreacted binding moieties that would
otherwise produce background by binding proteins in later flows;
flowing a buffer in vertical flow channels to passivate the
vertical flow channels; flowing a plurality of samples in vertical
flow channels to fill a corresponding pair of vertical flow
channels; circulating a fixed volume of the sample in the pair of
vertical flow channels to capture protein by the antibodies in
corresponding microchambers, the corresponding microchambers being
communicated to the pair of flow channels; flowing buffer in the
vertical flow channels to flush out the sample volume with any
unbound protein; flowing selected polyclonal antibodies in selected
horizontal flow chambers to build up an immunostack in the
microchambers; flowing buffer in the horizontal flow channels to
remove unattached polyclonal antibody; flowing fluorescently
labeled tags in the horizontal flow channels to tag the polyclonal
antibody; flowing a buffer in the horizontal flow channels to
remove excess unattached tags; and measuring fluorescence detection
in the microchambers.
16. The method of claim 15 where circulating a fixed volume of the
sample in the pair of vertical flow channels to capture protein by
the antibodies in corresponding microchambers comprises flowing the
fixed volume of the sample a closed path to maximize extraction of
the protein from the sample.
17. The method of claim 15 where prior to flowing a plurality of
samples in vertical flow channels to fill a corresponding pair of
vertical flow channels the method further comprises selectively
diluting selected ones of the samples with a standard buffer to
adjust the sample with a predetermined range of concentrations.
18. A method of performing a microfluidic assay comprising:
selectively flowing a plurality of antibodies in a plurality of
flow channels in communication with a plurality of microchambers in
a microfluidic matrix; selectively bonding selected antibodies to
binding moieties on a surface of the corresponding microchambers in
the microfluidic matrix; flowing a derivatization buffer in the
flow channels in the microfluidic matrix to remove unbound excess
protein and to passivate any unreacted binding moieties in the
microchambers that would otherwise produce background by binding
proteins in later flows; flowing a plurality of samples in flow
channels communicated to the microchambers in the microfluidic
matrix to fill a predetermined volume of the microfluidic matrix,
which predetermined volume at least includes the microchambers;
bonding a corresponding plurality of proteins to the selected
antibodies on the surface in the corresponding microchambers in the
microfluidic matrix; flowing buffer in the flow channels to flush
out the sample volume with any unbound protein from the
microchambers; and measuring bound protein in the plurality of
microchambers.
19. The method of claim 18 where bonding a corresponding plurality
of proteins to the selected antibodies on the surface in the
corresponding microchambers in the microfluidic matrix comprises
circulating a fixed volume of the sample in the flow channels to
capture protein by the antibodies in corresponding
microchambers.
20. The method of claim 18 further comprising: flowing a buffer in
flow channels to passivate the flow channels prior to bonding the
corresponding plurality of proteins to the selected antibodies on
the surface in the corresponding microchambers in the microfluidic
matrix; and flowing fluorescently labeled tags in the flow channels
to the plurality of microchambers to tag the sample and flowing a
buffer in the flow channels to remove excess unattached tags prior
to measuring bound protein in the plurality of microchambers.
Description
RELATED APPLICATIONS
[0001] The present application is related to U.S. Provisional
Patent Application Ser. No. 60/683,822, filed on May 23, 2005,
which is incorporated herein by reference and to which priority is
claimed pursuant to 35 USC 119.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention relates to the field of microfluidic circuits
and methods used to perform immunoassays.
[0005] 2. Description of the Prior Art
[0006] The ongoing revolution in biological sciences has generated
high hopes for the advent of true personalized/preventive medicine.
While the necessary biological tools are being developed at a fast
pace, it has become clear that their cost, operation, and
manufacturability are equally challenging issues that must be
solved before the new methods can be widely accepted in medical
practice. In the particular case of diagnostics, decentralized
"near-patient" or "point-of-care" testing (1) has attempted to
provide fast quantitative results at the bedside or in the clinic,
thereby decreasing hospital stays, eliminating transportation and
administrative expenses, and decreasing errors from mishandling and
miscommunication. While a few single-analyte systems (1) have been
developed (e.g., the now commonplace Glucometer.RTM.), the enormous
potential for decentralized testing remains untapped because the
vast majority of medical diagnostics is still conducted in clinical
laboratories and with the use of large equipment.
[0007] A way for ubiquitous near-patient and point-of-care testing
to reach fruition is for the current biological techniques to be
reduced from the macroscale to the microscale, in a multi-analyte
high-throughput format, preferably on handheld devices. In
particular, reducing immunoassays to microfluidic scales has been
extensively explored in recent years. Many approaches have been
proposed, involving glass, TiO.sub.2, silicon, and silicone
devices, but none possesses all of the desirable qualities: (i)
capability to measure multiple antigens and samples per device,
(ii) industrially feasible fabrication, (iii) parsimony of sample
and reagents, (iv) adequate sensitivity and specificity, and (v)
good reliability and reproducibility.
[0008] High kit and instrumentation costs dictate centralization of
measurements in large clinical or reference laboratories, resulting
in transportation and batch delays of up to 14 days between the
phlebotomist appointment and the final availability of the test
results. Such delays and the macroscale of samples and reagents
drive up the expenses in today's fast-paced and expensive
healthcare environment.
BRIEF SUMMARY OF THE INVENTION
[0009] The illustrated embodiment of the invention is a
high-throughput multi-antigen microfluidic fluorescence immunoassay
system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up
to 5 tests for each of 10 samples. In this particular study system,
the specificity of detection was demonstrated, and calibration
curves were produced for C-reactive protein (CRP),
prostate-specific antigen (PSA), ferritin, and vascular endothelial
growth factor (VEGF). The measurements show sensitivity at and
below clinically normal levels (with a signal-to-noise ratio >8
at as low as 10 pM antigen concentration). The chip uses 100 nL per
sample for all tests. The developed system is an important step
toward derivative immunoassay applications in scientific research
and "point-of-care" testing in medicine.
[0010] The circuits or chips of the illustrated embodiment
multiplex an immunoassay scheme to allow five simultaneous tests
for each of ten samples. Micromechanical valves direct the
pressure-driven reagent flow as desired along a network of 10
.mu.m-tall flow channels. "Four-way" valving at each intersection
in the test matrix forms a capture microchamber at the intersection
of crossing flow channels within which capture microchamber the
immunoassay stack is built for a particular sample-test
combination.
[0011] The basic steps of the assay are as follows. First,
monoclonal antibodies flowing in horizontal flow channels from
antigen inputs to derivitization exhausts respectively bind to the
epoxide coating of the floor of microchamber. Appropriate ones of
the valves are opened and closed by providing pressure to a
selected one of control ports to isolate the antibody flow from
unused portions of the flow channels of chip. It should be noted
that the manner of actuation of valves is non-exclusive of other
possibilities, e.g. electric actuation; thus, the approach is not
limited to pneumatically controlled valves.
[0012] Second, by providing pressure to a selected one of control
ports to again isolate flow from unused portions of the flow
channels of chip, a Tris buffer flowing in horizontal flow channels
is used to flushed from a buffer input to derivitization exhausts
and from a sample input flowing in vertical flow channels to sample
exhausts to inactivate remaining epoxide groups which have not
bound to an antigen. Other surface chemistry bindings are also
possible, e.g. carboxylate surface binding amino groups in the
presence of catalyst, and thus the approach is not limited to
epoxide chemistry. In fact, the same is in principle doable on PDMS
surfaces, in view of ref. Kartalov et al., Nucleic Acids Research
(2004).
[0013] Third, samples are fed in vertical flow channels in parallel
from sample inputs to sample exhausts. Again pressure is provided
to a selected one of control ports to again isolate flow from the
portions of the flow channels of chip not used for this purpose.
But valve actuation could also be accomplished by other means to
the same result.
[0014] Fourth, the sample which is then in place in the central
portion of matrix is pumped along closed paths or coliseums through
the capture microchambers. The coliseum communicates with two
microchambers and has a total volume of 10 nL which allows the
captured sample to be volumetrically quantized, which is
advantageous, if not essential, to making a practical quantified
measurement of the sample analyte. It is to be understood that the
arrangement of coliseum is shown by way of example only and that
other configurations can be employed without departing from the
scope and spirit of the invention. For example, a single or more
than two microchambers may be communicated to the coliseum, the
coliseum may be provided with a different pumping arrangement or
volume, and/or an oscillating flow pattern might be employed
instead of a circulating pattern.
[0015] Fifth, biotinylated polyclonal antibodies fed from antibody
inputs flow along horizontal flow channels to derivitization
exhausts to complete the immunostacks in the microchambers. Labeled
streptavidin fed from an antibody input to sample exhausts binds to
the immunostacks. It is to be understood that the operation
building the immunostack will be varied as may be required by the
scheme of the immunoassay employed. Also the manner of labeling is
not limited to the one described, as, for example, direct coupling
of fluorescence tags to polyclonal antibodies is possible with
commercial kits (e.g. Pierce), thereby circumventing the use of
labeled streptavidin. Again pressure is provided to a selected one
of control ports to again isolate flow from the portions of the
flow channels of chip not used for this purpose. Again the approach
is not limited to pneumatically actuated valves.
[0016] Sixth, a conventional fluorescence readout is performed.
Where the detection mechanism is not fluorescent, the detection
step and the detector used will be modified accordingly. The
detected fluorescence signal quantifies the captured antigens. In a
fluorescence readout a microscope and CCD camera employed, or a
micro-CCD array without a microscope, a film plate or any other
light detection means can be substituted.
[0017] The illustrated embodiment of the invention can thus be
characterized as a microfluidic assay apparatus comprising a
matrix, a plurality of sample/buffer flow channels defined in the
matrix, a plurality of antibody/buffer flow channels defined in the
matrix and intersecting the plurality of sample/buffer flow
channels, a corresponding plurality of selectively controllable,
valved capture microchambers, the capture microchamber being
defined at each intersection of the plurality of sample/buffer flow
channels and the plurality of antibody/buffer flow channels, means
for collecting a protein in the plurality of capture microchambers,
and means for detecting the plurality of collected proteins in the
capture microchambers.
[0018] The means for detecting the plurality of collected proteins
in the capture microchambers comprises means for quantifying the
concentration of the protein, which is collected in the capture
microchambers.
[0019] The means for detecting the plurality of collected proteins
in the capture microchambers comprises means for qualitatively
identifying the protein, which is collected in the capture
microchambers.
[0020] The means for collecting the protein in the plurality of
capture microchambers comprises means for simultaneously collecting
a plurality of different kinds of proteins in corresponding
different capture microchambers.
[0021] The plurality of sample/buffer flow channels are arranged
and configured to simultaneously receive a plurality of different
samples.
[0022] The matrix is comprised of a selectively epoxide coated
substrate and at least one PDMS layer disposed on the epoxide
coated substrate. Each of the selectively controllable, valved
capture microchambers are defined in the at least one PDMS layer
and comprise at least one push-down or push-up valve to control
flow into or out of the capture microchamber.
[0023] In the illustrated embodiment the means for simultaneously
collecting a plurality of different kinds of proteins in
corresponding different capture microchambers comprises a plurality
of antigens that are blood analytes, but the approach is not
limited to blood analytes only.
[0024] The microfluidic assay apparatus of claim 1 where the
plurality of antigens are selectively attached to the substrate by
means of selectively accessing epoxide coated substrate
surfaces.
[0025] One or more of the plurality of sample/buffer flow channels
are selectively coupled through selective communication with one or
more controllable, valved capture microchambers to form a
circulation or oscillation path of fixed volume and further
comprising a pump included in the path to circulate or oscillate
fluid in the path for a predetermined interval to increase
collection of the protein in the at least two capture
microchambers.
[0026] The plurality of capture microchambers are selectively sized
to provide a capture surface, which is scaled according to an
expected concentration of protein. Smaller capture surfaces are
used for lower expected concentrations, to reduce total integrated
background and thus improve signal-to-noise. Larger capture
surfaces are used for higher expected concentrations, to allow the
capture of more protein without surface saturation.
[0027] The microfluidic assay apparatus further comprises means for
diluting a sample with a predetermined amount of buffer to adjust
the sample concentration into an acceptable range of measurement
within the microchambers.
[0028] The illustrated embodiment of the invention similarly can be
characterized as a method of performing a microfluidic assay
comprising the steps of selectively flowing selected monoclonal
antibodies in a plurality of horizontal flow channels in a
microfluidic, optically transparent, biologically inert matrix,
selectively bonding selected monoclonal antibodies to binding
moieties on a surface in a corresponding microchambers in the
microfluidic matrix, flowing a derivatization buffer in the
horizontal flow channels to remove unbound excess protein and to
passivate any unreacted binding moieties that would otherwise
produce background by binding proteins in later flows, flowing a
buffer in vertical flow channels to passivate the vertical flow
channels, flowing a plurality of samples in vertical flow channels
to fill a corresponding pair of vertical flow channels, circulating
a fixed volume of the sample in the pair of vertical flow channels
to capture protein by the antibodies in corresponding
microchambers, the corresponding microchambers being communicated
to the pair of flow channels, flowing buffer in the vertical flow
channels to flush out the sample volume with any unbound protein,
flowing selected polyclonal antibodies in selected horizontal flow
chambers to build up an immunostack in the microchambers, flowing
buffer in the horizontal flow channels to remove unattached
polyclonal antibody, flowing fluorescently labeled tags in the
horizontal flow channels to tag the polyclonal antibody, flowing a
buffer in the horizontal flow channels to remove excess unattached
tags, and measuring fluorescence detection in the microchambers.
Here and henceforth, "horizontal" and "vertical" refer to the
orientation of the sets of channels as shown on the attached
figures, rather than relative orientation with respect to gravity
vector in the physical device.
[0029] The step of circulating a fixed volume of the sample in the
pair of vertical flow channels to capture protein by the antibodies
in corresponding microchambers comprises flowing the fixed volume
of the sample along a closed path to maximize extraction of the
protein from the sample, by exposing the same capture surface to
all sections of the volume one or multiple times.
[0030] Prior to flowing a plurality of samples in vertical flow
channels to fill a corresponding pair of vertical flow channels,
the method further comprises selectively diluting selected ones of
the samples with a standard buffer to adjust the sample with a
predetermined range of concentrations.
[0031] Yet another embodiment of the invention is a method for
performing a microfluidic assay comprising the steps of selectively
flowing a plurality of antibodies in a plurality of flow channels
in communication with a plurality of microchambers in a
microfluidic matrix, selectively bonding selected antibodies to
binding moieties on a surface of the corresponding microchambers in
the microfluidic matrix, flowing a derivatization buffer in the
flow channels in the microfluidic matrix to remove unbound excess
protein and to passivate any unreacted binding moieties in the
microchambers that would otherwise produce background by binding
proteins in later flows, flowing a plurality of samples in flow
channels communicated to the microchambers in the microfluidic
matrix to fill a predetermined volume of the microfluidic matrix,
which predetermined volume at least includes the microchambers,
bonding a corresponding plurality of proteins to the selected
antibodies on the surface in the corresponding microchambers in the
microfluidic matrix, flowing buffer in the flow channels to flush
out the sample volume with any unbound protein from the
microchambers, and measuring bound protein in the plurality of
microchambers.
[0032] In this last embodiment the step of bonding a corresponding
plurality of proteins to the selected antibodies on the surface in
the corresponding microchambers in the microfluidic matrix
comprises circulating a fixed volume of the sample in the flow
channels to capture protein by the antibodies in corresponding
microchambers.
[0033] In this same last embodiment the method further comprises
the steps of flowing a buffer in flow channels to passivate the
flow channels prior to bonding the corresponding plurality of
proteins to the selected antibodies on the surface in the
corresponding microchambers in the microfluidic matrix, and flowing
fluorescently labeled tags in the flow channels to the plurality of
microchambers to tag the sample and flowing a buffer in the flow
channels to remove excess unattached tags prior to measuring bound
protein in the plurality of microchambers.
[0034] While the apparatus and method has or will be described for
the sake of grammatical fluidity with functional explanations, it
is to be expressly understood that the claims, unless expressly
formulated under 35 USC 112, are not to be construed as necessarily
limited in any way by the construction of "means" or "steps"
limitations, but are to be accorded the full scope of the meaning
and equivalents of the definition provided by the claims under the
judicial doctrine of equivalents, and in the case where the claims
are expressly formulated under 35 USC 112 are to be accorded full
statutory equivalents under 35 USC 112. The invention can be better
visualized by turning now to the following drawings wherein like
elements are referenced by like numerals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a side cross sectional view of a push-down valve
used in the microfluidic circuit of the invention.
[0036] FIG. 2 is a top plan view of the microfluidic circuit of the
illustrated embodiment.
[0037] FIG. 3a is a diagram of a top plan view of a full sized
microchamber used in the microfluidic circuit of the invention.
[0038] FIG. 3b is a diagram of a top plan view of a reduced sized
microchamber used in the microfluidic circuit of the invention.
[0039] FIG. 4 is a diagram of an immunoassay stack built up at the
microchambers in the microfluidic circuit of the invention.
[0040] FIG. 5 is a diagram in top plan view of a layout of a
circulating flow path or coliseum in the microfluidic circuit of
the invention.
[0041] FIG. 6 is a microphotograph of the fluorescence image of a
microchamber. for a VEGF test of a 0.3 nM sample produced in the
microfluidic circuit of the invention.
[0042] FIG. 7 is a bar graph of the specificity for five four
selected human blood antigens (CRP, VEGF, PSA, ferritin) and a BSA
"negative" control sample, using five corresponding microchambers
in the microfluidic circuit of the invention.
[0043] FIGS. 8a-8d are graphs of the net output signal read from
microchambers in the microfluidic circuit of the invention as a
function of antigen concentration for PSA, VEGF, CRP and Ferritin
in simple solutions (PBS buffer with 0.1% BSA). Twenty samples of
known concentrations were tested in two experiments to produce
calibration curves for four antigens: PSA, prostate-specific
antigen; VEGF, vascular endothelial growth factor; and CRP,
C-reactive protein. These blood analytes are related to
inflammation, prostate cancer, long-term iron buildup, and cancer,
respectively. The system demonstrated sensitivity at the clinically
relevant abundances (with a signal-to-noise ratio >8 at as low
as 10 pM) while using only 100 nL per sample for all tests and only
300 nL of antibody per test for all samples.
[0044] FIGS. 9a-9d are graphs of calibrations showing the net
signal output vs a reference concentration as measured by the
illustrated embodiment in human plasma for VEGF, PSA, Ferritin and
Thyroglobulin respectively.
[0045] FIGS. 10a-10d are graphs of calibrations showing the net
signal output vs a reference concentration as measured by the
illustrated embodiment in human serum for VEGF, PSA, Ferritin and
CRP respectively.
[0046] The invention and its various embodiments can now be better
understood by turning to the following detailed description of the
preferred embodiments which are presented as illustrated examples
of the invention defined in the claims. It is expressly understood
that the invention as defined by the claims may be broader than the
illustrated embodiments described below.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0047] The illustrated embodiment of the invention is a
high-throughput multi-antigen high-specificity high-sensitivity
reproducible polydimethylsiloxane (PDMS) microfluidic system 10 for
quantifying four representative blood analytes 12 at the clinically
relevant levels. It is expressly to be understood that the
invention could be realized in different systems for quantifying or
identifying different numbers of different analytes and in
different types of biological samples other than blood, such as
urine, spinal fluid, vaginal secretions, perspiration, saliva,
synovial fluid, cerebral fluid, ocular fluid, biopsy samples, and
many other tissue sample types where the nanoliter sized sample of
the invention make testing possible and practical for the first
time. The illustrated embodiment is set forth here only for
illustration and concreteness of example.
[0048] An active microfluidic matrix 14 utilizes arrays of
integrated micromechanical or microhydraulically actuated valves 16
to direct pressure-driven flow and multiplex analyte samples 12
with immunoassay reagents 18. Enzyme-linked immunosorbent assay
(ELISA)-like fluorescence immunostacks 20 are formed in the
microchambers 22 at the intersections of sample channels 24 and
reagent channels 26. The fluorescence signals from these
microchambers 22 quantify and identify the captured antigens 28.
However, the detection mechanism and the corresponding detector to
read the assay may assume many different equivalent forms, such as
the use of direct fluorophores instead of fluorophores tags,
fluorophores by proxy, chemiluminescence, quantum dots, radioactive
tags and the like. Any means by which a radiative quantification
can be obtained can be substituted.
[0049] The 100-chamber system 10 of the illustrated embodiment
conducts five tests for each of ten samples 12 with two replicates
per sample-test combination. The number of samples can be
significantly increased by extending the size of the matrix 14
according to the teachings of the invention by exploiting the known
capabilities of PDMS microfluidic technology. In the illustrated
embodiment we chose blood analytes 12 for initial validation of the
system 10, because blood tests represent one of the most common
examples of routine use of immunoassays. However, it is to
expressly understood that any kind of analyte, biological or
otherwise, could be subjected to the system 10 and methodology of
the invention.
[0050] The current standard clinical macrofluidic technology is
typically based on an enzyme-linked immunosorbent assay (ELISA) and
in practice requires 0.5 to 2 mL of sample per test per patient. By
contrast with the high sample volumes required by conventional
ELISA, the system described here uses only 100 nL of sample for ten
tests, while simultaneously measuring CRP, PSA, ferritin, and VEGF
within the clinically significant range. The system also uses only
300 nL of antibodies (as low as 0.8 ng) per assay to measure all
ten samples. Therefore, the microfluidic miniaturization of
immunoassays described here paves the way to efficient and portable
hand-held devices to be used by the attending medical staff in the
field or in the clinic at the time of sampling.
[0051] FIG. 1 is a diagram showing a side cross sectional view of a
portion of matrix 14, which in the illustrated embodiment is built
up on a glass substrate 30, which is selectively coated with
epoxide 74 or have other adhesion promotion substances coated on
its upper surface to promote molecular attachment of antibodies 56
to substrate 30 as discussed in connection with FIG. 4. A thin
molded layer 32 of PDMS is then disposed, attached, molded onto a
thicker control layer 36. Layer 32 is the flow layer in which flow
channels 24 are defined or molded as described below. Control layer
36 and flow layer 32 are then bound to epoxide-coated substrate 30.
PDMS is preferred because it is optically transparent in the
visible spectrum, is biologically inert, and can be easily molded
or formed using conventional soft lithography. Layer 32 may
typically be in the range of 26 to 49 .mu.m thickness.
[0052] A plurality of deformable valve chambers 34 are defined in
layer 32, which can be employed as hydraulically actuated push-down
or push-up valves 38, depending on the structure chosen, or
combined to function as pumps 40 as described in connection with
FIG. 5. FIG. 1 illustrates a single push down valve 38. Valve
chamber or valve microchamber 34 will typically be defined in a
flow channel 24, best shown in the views of FIGS. 3a and 3b. A
thicker control layer 36 of PDMS is disposed on layer 32 into which
is defined a pressure chamber 42 communicated by a control channel
92 to a control port 46. Hydraulic fluid is pumped by an exterior
pump or pumps, not shown, into pressure chamber 42 thereby forcing
down the thinner portion 48 of layer 32 overlying the pushdown
valve 38 of which valve chamber 34 is part. By this means, any flow
channel 24 in which valve 38 is defined can be selectively shut or
closed off by selective pressurization of pressure chamber 42,
thereby pushing down portion 48 of layer 32 against portion 50 of
substrate 30.
[0053] In the illustrated embodiment, a microfluidic circuit 52 in
matrix 14 is shown in top plan view of FIG. 2 and is formed by the
method described below. Selected positions 54 on the treated upper
surface of substrate 30 are provided with selected reagents
according to the assay which is to be performed at that site.
Positions 55 will underlie intersections of overlying flow channels
24 and comprise the floor of capture microchambers 54. FIGS. 3a and
3b illustrate a top plan view two types of orthogonal intersections
of two channels 24 in circuit 52. The site of the intersection
defines the selected position 55 and the corresponding valved
capture microchamber 54, where a reagent will be selectively
provided on substrate 30 by means of selective coating of substrate
30 by an epoxide or other molecular adhesion agent at positions 55
and where a test will be performed.
[0054] Before considering the fabrication of circuit 52 of FIG. 2
consider first the basic scheme by which immunoassays are made in
circuit 52. In sandwich immunoassays, a monoclonal antibody 56 as
diagrammed in FIG. 4, specific to the target analyte (antigen 28),
is bound to a position or surface 55 in capture microchamber 54 by
molecular bonding to an epoxide coating selective provided on
surface 55. Different antigens 28 are attached by selectively
flowing a solution carrying the chosen antigen 28 through the
corresponding horizontal flow channel 24 from one of the antigen
ports 88(1)-88(5) described in connection with FIG. 2. Next, the
sample 12 is put in contact with surface 54 by selectively flowing
a solution carrying the sample 12 through the corresponding
vertical flow channel 24 from one of the sample ports 82(1)-82(10)
described in connection with FIG. 2, whereby the antibody 56
captures the contained antigen 28. Then, a labeled polyclonal
antibody 58 is provided by selectively flowing a solution carrying
the chosen polyclonal antibody 58 through the corresponding
horizontal flow channel 24 from one of the antigen ports
88(1)-88(5) described in connection with FIG. 2, which attaches to
the antigen 28 to complete the immunostack 20. The label (e.g., a
linked enzyme 60 creating fluorescent product or a fluorophore 62
bound to the polyclonal antibody 58) generates a light signal using
conventional fluoroscopic detection techniques that is compared
with a standard to quantify the captured antigen 28. FIG. 6 is a
microphotograph which shows a typical fluorescence image from
microchamber 54 formed by 20 .mu.m-wide channels for a VEGF test of
0.3 nM sample.
[0055] Consider now a listing of reagents and materials used in
chip fabrication in the illustrated embodiment. The materials used
in chip fabrication included Hexamethyldisilazane (HMDS) adhesion
promoter applied to substrate 30 obtained from ShinEtsuMicroSi
(Phoenix, Ariz., USA). The photoresist used in chip fabrication
(Shipley SJR 5740) was obtained from MicroChem (Newton, Mass.,
USA). Tetramethyl-chlorosilane (TMCS) was obtained from Sigma (St.
Louis, Mo., USA). PDMS Sylgard 184 was obtained from Dow Corning
(Midland, Mich., USA). Arraylt.RTM. SuperEpoxide SME slides for
substrate 30 were obtained from TeleChem International (Sunnyvale,
Calif., USA). It must of course be understood that this list of
materials is not a limitation on the kinds of materials that can be
used to fabricate circuits 52.
[0056] Turn now to the antibodies and antigens relevant to the
illustrated embodiment. PSA antigen, monoclonal PSA antibody,
ferritin antigen, monoclonal ferritin antibody, and monoclonal CRP
antibody were procured from Fitzgerald Industries (Concord, Mass.,
USA); VEGF antigen and antibodies and biotinylated CRP antibody
from R&D Systems (Minneapolis, Minn., USA); PSA biotinylated
antibody from Lab Vision (Fremont, Calif., USA); ferritin
biotinylated antibody from U.S. Biological (Swampscott, Mass.,
USA); and CRP antigen from EMD Biosciences (Calbiochem.RTM.; San
Diego, Calif., USA). Again, it must be explicitly understood that
this list of antibodies and antigens is not a limitation on the
kinds of biological compounds that can be used or tested in
circuits 52.
[0057] Finally, consider the fluorescent probes and buffers used in
the illustrated embodiment. Streptavidin Alexa Fluor.RTM. 555 was
supplied by Invitrogen (Molecular Probes.TM.; Carlsbad, Calif.,
USA). Lyophilized commercial antigens and antibodies were
reconstituted in phosphate-buffered saline (PBS) 1.times. buffer
from Irvine Scientific (Santa Ana, Calif., USA), pH 7.5. Bovine
serum albumin (BSA) was added to the same to produce the PBS 0.1%
BSA buffer. The passivation buffer was 10 mM Tris, 10 mM NaCl, pH
8.0, made from powdered Tris and NaCl (both from Sigma). It must be
explicitly understood that this list of fluorescent probes and
buffers is not a limitation on the kinds of probes and buffers that
can be used or tested in circuits 52.
[0058] Consider now the general method of making a mold for a
circuit 52 such as that shown in FIG. 2. PDMS microfluidic chips 52
with integrated micro-mechanical valves 38 were built using
conventional soft lithography with the following modifications.
Silicon wafers used as a negative mold were exposed to HMDS vapor
for 3 min. The silicon wafers were coated with Photoresist SPR
220-7 by spinning at 2000 rpm for 60 s on a WS-400A-GNPP/LITE
spincoater (Laurell Technologies, North Wales, Pa., USA). The
silicon wafers were baked at 105.degree. C. for 90 s on a hotplate.
UV exposure through black-and-white transparency masks was
performed for 1.75 min on a Karl Suss MJB3 mask aligner (Karl Suss
America, Waterbury, Vt., USA). The molds were then developed for 2
min in 100% MicroChem 319 developer (MicroChem). Flow layer molds
were baked at 140.degree. C. for 15 min on a hotplate to melt and
round the flow channels 24. Molds were characterized on an
Alpha-Step 500 (KLA-Tencor, Mountain View, Calif., USA). Channel
height was between 9 and 10 .mu.m. The control channel 92 profile
was rectangular, while the flow channel 24 profile was parabolic.
Except for the height measurements, the mold fabrication was
conducted in a Class 10,000 clean room.
[0059] The molds were exposed to TMCS vapor for 3 min. PDMS in 5:1
and 20:1 ratios were mixed and degassed using an HM-501 hybrid
mixer and cups from Keyence (Long Beach, Calif., USA). Thirty-five
grams of the 5:1 were poured onto the control mold used to make the
control layer 36 in a plastic Petri dish wrapped with aluminum
foil. Five grams of the 20:1 were spun over the flow mold at 1500
rpm for 60 s using a P6700 spincoater from Specialty Coating
Systems (Indianapolis, Ind., USA). Both were baked in an 80.degree.
C. oven for 30 min. The control layer 36 was taken off its mold and
cut into respective chips pieces or portions. Control line ports 46
were punched using a 20-gauge Intramedic.TM. Luer-Stub adapter (BD
Biosciences, Franklin Lakes, N.J., USA). Control layer 36 pieces
were washed with ethanol, blown dry with filtered air or nitrogen,
and aligned on top of the flow layer 32 under a stereoscope. The
result was baked in an 80.degree. C. oven for 1 h. Chip pieces were
then cut out and peeled off the flow layer 32 mold. Flow line ports
68(i), 70(i), 72, 78, 80(i), 82(i), 88(i), and 90 shown in FIG. 2
were punched with the 20-gauge Luer-stub adapter. Chip pieces were
then washed in ethanol and blown dry before binding to the epoxide
glass slides 30. The assembled chips 52 of FIG. 1 underwent a final
bake overnight in an 80.degree. C. oven.
[0060] To bench test the assembled chip 52 an inverted Olympus IX50
microscope (Olympus America, Melville, N.Y., USA) was equipped with
a mercury lamp (HBO.RTM. 103 W/2; Osram, Munich, Germany), an
Olympus Plan 10.times. objective [numerical aperture (NA) 0.25], a
long-distance Olympus SLCPlanFI 40.times. objective (NA 0.55), a
cooled charge-coupled device (CCD) camera (Model SBIG ST-71; Santa
Barbara Instrument Group, Santa Barbara, Calif., USA), and a
fluorescence filter set (excitation: D540/25, dichroic 565 DCLP;
emission: D605/55) from Chroma Technology (Brattleboro, Vt., USA).
We then plugged 23-gauge steel tubes from New England Small Tube
(Litchfield, N.H., USA) into the chip's control channel ports 46
described below. Their other ends were connected through Tygon.RTM.
tubing (Cole-Parmer, Vernon Hills, Ill., USA) to Lee-valve arrays
(Fluidigm, San Francisco, Calif., USA) operated by LabView software
on a personal computer. The same types of steel tubes and Tygon
plumbing were used to supply reagents to the chip's flow channel
ports 66 described below. It is to be understood that the
illustrated embodiment is a bench prototype and that the elements
of a control system for providing pressurized control fluid,
antigens, buffers, samples and the like will be modified from that
disclosed to be optimized and miniaturized in the commercial
production system according to conventional engineering design
principles.
[0061] The immunoassay and the fabrication of chip 52 having been
described, it is possible now to consider the implementation of
chip 52 in matrix 14 according to the invention as shown in the
example of FIG. 2. The microfluidic immunoassays chip 52 of FIG. 2
includes in the illustrated embodiment a 100-chamber, 22.times.35
mm PDMS chip 52 bound to an epoxide slide 30 to simultaneously
perform five tests each of ten samples, with two chambers 54 per
sample-test combination. Control channels 92 are shown in dark
outline and convey hydraulic pressure to open and close microvalves
38 that direct pressure-driven feeds of reagents along flow
channels 24 according to the valve action as described in
connection with in FIG. 1. Each intersection of flow channels 24 in
the central test matrix 14 forms a microchamber 54 where an
immunostack 20 is constructed as described above. The flow channels
are described below as comprised of horizontal and vertical flow
channels 24 and are shown as such in FIGS. 2, 3a and 3b. However,
it is to be understood that orientation with respect to any defined
direction is immaterial, that the intersections of flow channels 24
need not be orthogonal, but may be skewed, and in fact
microchambers 54 and channels 24 need not be rasterized in a matrix
array, but topologically organized in any arrangement consistent
with the teachings of the invention. Nevertheless, it is the
preferred embodiment to have channel and microchamber layout which
is both rasterized and orthogonal for ease of manufacture and
scaling the matrix size or number up or down as desired.
[0062] Turn now to the layout of circuit or chip 52 of the
illustrated embodiment as shown in plan view in FIG. 2 and consider
the steps summarized above taken in a measurement or assay in more
detail. In a typical measurement, monoclonal antibodies 56 flow in
horizontal flow channels 24 from derivatization inputs 68(1)-68(5)
to derivatization exhausts 70(1)-70(5) in FIG. 2. The antibodies 56
covalently bond to the epoxide floor 54 of the microchannels 24,
producing the first layer of the immunostack 20 in FIG. 4. Tris
buffer 76 flowing from derivatization buffer input 72 flows in
horizontal flow channels 24 to derivatization exhausts 70(1)-70(5)
to remove unbound excess protein and passivates any unreacted
epoxide moieties 74 that would otherwise produce background by
binding protein in later feeds. Next, Tris buffer 76 flows from
samples buffer input 78 in vertical flow channels 24 to samples
exhausts 80(1)-80(10) to passivate the rest of the microchannels
24.
[0063] As samples 12 flow in parallel in vertical flow channels 24
from sample inputs 82(1)-82(10) to sample exhausts 80(1)-80(10),
each sample 12 fills a corresponding pair of microchannels 24. When
the appropriate valves 38 are closed, each such pair of
microchannels 24 forms a closed path, called here a coliseum 84,
that traps 10 nL of the respective sample 12 as shown in better
view in FIG. 5. Then, an array of peristaltic micropumps 86(i),
which are three cyclically driven valves as shown in FIG. 1, drive
each trapped volume around its coliseum 84, with a lap time of 20
s. A cyclical application of pressure is provided to micropumps
86(i) through control flow channels 92 from three corresponding
control ports 46 provided from an exterior pump and controller (not
shown). Within each coliseum 84, each antigen 28 is captured in its
respective microchamber 54, as determined by the first layer of the
immunostack 20. The same sample 12 is allowed to run multiple laps
(typically 10) to maximize extraction of the antigen 28 from the
sample 12.
[0064] FIG. 5 is a functional diagram of an individual coliseum 84.
Here control channels 92 and valves 38 are drawn in dark outline
and flow channels 24 in lighter outline. Comb like valve arrays
86(1)-86(3) enclose a pair of immunoassay chambers 54 for each of
five tests. Valve arrays 86(1)-86(3) pump the sample 12 in a circle
along the coliseum 84 [e.g., clockwise for actuation order (86(1),
86(2), 86(3)] with a lap time of 20 s. Again pressure is provided
to a selected one of control ports 46 to again isolate flow from
the portions of the flow channels 24 of chip 52 not used for this
purpose.
[0065] After harvesting, buffer 76 from samples buffer port 78 flow
in vertical flow channels to sample exhaust ports 80(1)-80 (10) to
flush out the sample volume. Parallel feeds of biotinylated
antibodies 58 from antibody inputs 88(1)-88(5) flow in horizontal
flow channels 24 to derivatization exhausts 70(1)-70(5)
respectively to build up the third layers of the immunostacks 20 in
each microchamber 54. Buffer 76 from derivatization buffer input 72
flow in horizontal flow channels 24 to derivatization exhausts
70(1)-70(5) to remove unattached antibody 58. Fluorescently labeled
streptavidin 60 in PBS buffer flows from streptavidin input 90 in
horizontal flow channels 24 to derivatization exhausts 70(1)-70(5).
Then, buffer from derivatization buffer input 72 flows in
horizontal flow channels 24 to derivatization exhausts 70(1)-70(5)
to remove unattached excess. All valves 38 are then closed, and
fluorescence detection is conducted at each microchamber 54 using
an inverted optical microscope and an inexpensive, cooled CCD
camera or other detection means, which produces an image as shown
in FIG. 6 of each chamber 54.
[0066] Chip 52 now having been described and its fabrication
disclosed, consider the performance of the illustrated embodiment
with respect to blood protein assays. Blood proteins were chosen to
validate the system because blood tests are one of the most common
and clinically important applications of immunoassays. In
particular, CRP, PSA, ferritin, and VEGF were selected due to their
significance in medical diagnostics, the wide concentration range
spanned by their clinically normal levels, and the commercial
availability of well-validated antigens and antibodies.
[0067] To test the specificity of the system, we processed one load
of 10 nL for each of four samples, each containing 20 nM of one of
the antigens in PBS 0.1% BSA, in a chip with 100 .mu.m-wide
channels (approximately 50,000 .mu.m.sup.2 per microchamber 54).
Because every test lane intersects every coliseum 84 in a pair of
microchambers 54, the fluorescence signals of each such pair 54
were added to produce the signal for the respective sample-test
combination. After normalizing for area, we divided each signal by
the fluorescent background of the particular test as measured in
regions unexposed to antigen.
[0068] The results are graphed in FIG. 7. Samples 12 each
containing 20 nM of a single antigen, CRP, PSA, ferritin, and VEGF,
were fed in parallel into the test matrix 14. Each sample 12
produced significant signal above background only in the test
corresponding to the antigen contained in the sample 12. The BSA
control produced signal at the background level. The results showed
the specificity of measurement and the lack of crosstalk between
tests. Every sample 12 produced significant signal above background
only in the test chambers 54 corresponding to the antigen 28 it
contained. In addition, the presence of the antigens 28 does not
increase the background in the control case, where PBS 0.1% BSA
replaced the antibody feeds. These results demonstrate the
specificity of the system.
[0069] To test the sensitivity of the system, we ran 10 samples
against the same four tests but in devices with 20 .mu.m-wide
channels 24 at the intersections (approximately 2000 .mu.m.sup.2
per microchamber 54). One sample was a control containing no
antigen 28. Each of the other nine samples contained all antigens
at the same concentration, which was varied between 30 pM and 10 nM
from sample to sample, all in PBS 0.1% BSA. We processed 100 nL per
sample (10 loads of 10 nL). The signal for each sample-test was
extracted from fluorescence images of the chambers by subtracting
the local background for each image and adding the two such results
per sample-test combination. Then, for each test, the signal of the
control sample was subtracted from the signals of the other nine
samples to produce the final results for each test. To establish
reproducibility, the same experiment was repeated in another chip
with a new dilution of reagents. Also, the concentration range was
expanded (10 pM to 100 nM). Data analysis was conducted as
described above. The results were combined in a single plot per
test as graphed in FIGS. 8a-8d, including the clinically relevant
levels (www.labtestsonline.org). The data demonstrate the
reproducibility of results in the system.
[0070] The net signal for the lowest concentration (10 pM) for each
test was divided by the uncertainty of the respective control
signal to produce a measure of the observed signal to noise. The
results were 164 (CRP), 38 (PSA), 11 (ferritin), and 8 (VEGF).
[0071] The PSA test shows a linear calibration between 100 pM and
30 nM in FIG. 8a. This dynamic range includes the "gray zone" at
4.0-10.0 ng/mL (133-332 pM). The higher the concentration above the
"gray zone," the stronger the indication for prostate cancer.
Similarly, VEGF has a linear calibration between 10 pM and 10 nM in
FIG. 8b. This range includes the important cutoff at 25 ng/mL (0.1
nM), exceeding which is an indication for cancer.
[0072] The CRP test shows that the system is linear between 10 and
300 pM, after which the signal saturates in FIG. 8c. In this case,
the sensitivity is excessive since the clinically abnormal levels
are above 1.2 mg/dL (110 nM), indicating acute infection.
Similarly, the ferritin detection in FIG. 8d is sensitive within
and below the normal range of 30-300 ng/mL (60-630 pM) but
saturates above it, where long-term iron buildup is indicated.
[0073] The observed saturation for CRP and ferritin can be avoided
in a number of straightforward ways, producing chips 52 customized
to a particular set of tests. In such chips 52, the scarce-agent
tests would retain the smallest channels 24 for maximal sensitivity
as shown in FIG. 3b, while the abundant-agent tests would have
wider channels to increase capture area and thus raise the
saturation point as shown in FIG. 3a. Tests for ultra-abundant
agents (e.g., ceruloplasmin, normally at 21-50 mg/dL) would be
organized in another section of the chip 52 and would be preceded
by a dilution stage to reduce the concentration into the measurable
range. Because the dilution factor would be predetermined by the
device geometry, straightforward multiplication would yield the
correct final result.
[0074] FIGS. 9a-9b and 10a-10b show the human plasma and serum data
respectively which illustrates the system of the invention
functions correctly with real human serum/plasma samples and
moreover, reproduces the same calibration curves as with simple
solutions. In this case, the human samples were spiked with
commercially available pure antigens.
[0075] The parsimony of the system 10 is important in any
immunoassay application where sample 12 is costly or scarce. In
blood tests, the current practical requirement is 0.5-2 mL per
sample per test, necessitating drawing blood from the vein and
making common blood tests difficult for pediatric patients. In
contrast, the system presented here uses 100 nL of each sample for
all tests, thus enabling the development of portable apparatuses
conducting common blood tests by a finger prick.
[0076] Simultaneously, the system uses 300 nL (as low as 0.8 ng) of
antibody per sample-test combination. In contrast, the
state-of-the-art Elecsys.RTM. PSA kit from Roche Applied Science
(Indianapolis, Ind., USA) uses 200 ng per sample-test, or 250 times
more. The savings have direct consequences in modern healthcare and
biomedical research.
[0077] The produced calibration curves could be used as the
established dependences, which allow internal recalibrations to be
constructed within each measurement by running just a few reference
samples per device. This technique would eliminate systematic
sources of variation, such as quality and condition of reagents,
intensity of the illumination source, and differences in storage
and handling. Simultaneously, the results would be extended to more
complex media, such as human serum, plasma, spinal fluid, and
biopsy samples. Finally, the test matrix 14 could be expanded to
50.times.50 in commercial products.
[0078] The illustrated embodiment demonstrates the reduction of
immunoassays to a microfluidic high-throughput multi-antigen
format. The developed system 10 is an important step toward
derivative immunoassay applications in scientific research and
point-of-care testing in medicine.
[0079] Many alterations and modifications may be made by those
having ordinary skill in the art without departing from the spirit
and scope of the invention. Therefore, it must be understood that
the illustrated embodiment has been set forth only for the purposes
of example and that it should not be taken as limiting the
invention as defined by the following invention and its various
embodiments.
[0080] Thus, it can now be appreciated that the invention can be
reproduced in a portable, field usable unit which can provide
high-throughput, multi-antigen tests at low cost. Blood samples in
the volume of pin pricks can be utilized without the need for a
qualified phlebotomist. A multiple number of patients may be tested
using the same matrix with one sample input being taken from each
patient. Testing can be done at the surgery site on a continual
basis without need to delay or wait for conventional remote lab
testing. As the number of proteins discovered increases or their
significance to physiological function is discovered, the apparatus
of the invention can accommodate significant expansion in the
number of analytes testing, which increased numbers would overwhelm
and overrun conventional testing apparatus and procedures both in
terms of cost, time and feasibility. The specificity and
calibration of the methodology and apparatus of the invention
easily meets and exceeds current clinical standards and even
promises to raise those standards in many cases. At the same time,
the invention is noninvasive and utilizes conventional
immunoassays, thereby avoiding lengthy or complex FDA approvals.
The use of the apparatus is simple and inexpensive enough to
conveniently allow for patient self-monitoring in patients
suffering from diabetes, cancer, cardiovascular diseases or those
seeking hormonal or metabolic health, performance or fitness.
Finally, the invention lends itself to system integration so that
it can be practically and readily rendered a plurality of packages
and applications.
[0081] Therefore, it must be understood that the illustrated
embodiment has been set forth only for the purposes of example and
that it should not be taken as limiting the invention as defined by
the following claims. For example, notwithstanding the fact that
the elements of a claim are set forth below in a certain
combination, it must be expressly understood that the invention
includes other combinations of fewer, more or different elements,
which are disclosed in above even when not initially claimed in
such combinations. A teaching that two elements are combined in a
claimed combination is further to be understood as also allowing
for a claimed combination in which the two elements are not
combined with each other, but may be used alone or combined in
other combinations. The excision of any disclosed element of the
invention is explicitly contemplated as within the scope of the
invention.
[0082] The words used in this specification to describe the
invention and its various embodiments are to be understood not only
in the sense of their commonly defined meanings, but to include by
special definition in this specification structure, material or
acts beyond the scope of the commonly defined meanings. Thus if an
element can be understood in the context of this specification as
including more than one meaning, then its use in a claim must be
understood as being generic to all possible meanings supported by
the specification and by the word itself.
[0083] The definitions of the words or elements of the following
claims are, therefore, defined in this specification to include not
only the combination of elements which are literally set forth, but
all equivalent structure, material or acts for performing
substantially the same function in substantially the same way to
obtain substantially the same result. In this sense it is therefore
contemplated that an equivalent substitution of two or more
elements may be made for any one of the elements in the claims
below or that a single element may be substituted for two or more
elements in a claim. Although elements may be described above as
acting in certain combinations and even initially claimed as such,
it is to be expressly understood that one or more elements from a
claimed combination can in some cases be excised from the
combination and that the claimed combination may be directed to a
subcombination or variation of a subcombination.
[0084] Insubstantial changes from the claimed subject matter as
viewed by a person with ordinary skill in the art, now known or
later devised, are expressly contemplated as being equivalently
within the scope of the claims. Therefore, obvious substitutions
now or later known to one with ordinary skill in the art are
defined to be within the scope of the defined elements.
[0085] The claims are thus to be understood to include what is
specifically illustrated and described above, what is
conceptionally equivalent, what can be obviously substituted and
also what essentially incorporates the essential idea of the
invention.
* * * * *